Ex vivo animal or challenge model as method to measure protective immunity directed against parasites and vaccines shown to be protective in the method

ABSTRACT

The invention relates to an ex vivo animal or challenge model as a method to identify protective (recombinant) proteins and rapidly measure protective immunity in intestinal segments directed against parasites and vaccines directed against parasitic infections. The invention further relates to vaccines directed against infection with parasites, such as  Fasciola hepatica , which vaccines contain protective (recombinant) proteins identified and shown to be protective in studies using the ex vivo model. The invention further relates to protective (recombinant) proteins obtained from newly excysted juveniles (NEJ) of  Fasciola hepatica . The protective (recombinant) protein corresponding to an NEJ protein has an apparent molecular weight of 32 kD and an N-terminal amino acid sequence comprising the sequence XXDVSWPFWDRMYNY (SEQ ID NO:1).

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.10/382,479, filed Mar. 6, 2003 now abandoned, which is a continuation ofU.S. patent application Ser. No. 09/381,122, filed Dec. 23, 1999, nowU.S. Pat. No. 6,551,594, issued Apr. 22, 2003, which claims priorityfrom PCT Patent Application No. PCT/NL98/00146, filed Mar. 11, 1998, andpublished, in English, on Sep. 17, 1998, as WO 98/40497, the entirecontents of each of which are incorporated herein by this reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to biotechnology generally and, morespecifically, to an ex vivo animal or challenge model as a method tomeasure protective circuitry directed against parasites and vaccinesshown to be protective in the method.

2. State of the Art

Only a few vaccines against parasites are commercially available. Mostof these vaccines are based on attenuated live parasites that inducenatural, protective immunity and cause less severe pathological damage.These parasite vaccines include one directed against Dictyocaulusviviparus (e.g., Dictol, Glaxo), undoubtedly the most successfulanti-parasite vaccine, and analogous therewith a vaccine againstDictyocaulus filaria, the lung worm in sheep (Sharma et al. 1988). Thesevaccines are based on live but irradiated third-stage larvae (Peacockand Pointer 1980). Another attenuated vaccine is directed against thehookworm Ancylostoma caninum in dogs. However, this vaccine has beenmarketed only for a short time in the USA; marketing was discontinuedbecause the American veterinary profession did not accept this livevaccine (Urquhart 1980). An attenuated vaccine against Babesia bovis hasbeen in use for nearly a century in Australia (Purnell 1980) and a deadvaccine based on metabolic products named “Pirodog” is used to vaccinatedogs against B. canis (Moreau 1986).

Vaccination trials in sheep with a recombinant vaccine against the tapeworm Taenia ovis (Johnson et al. 1989) and the concealed antigen H11from Haemonchus contortus (Newton 1995, review) have been performedsuccessfully. A trial with the SPf66 malaria vaccine in Africa hasrecently been completed. The efficiency against clinical malaria inareas of high transmission was 31% and the product appeared to be safe.However, because it is not fully understood how SPf66 mediatesprotection, the development of improved vaccines is hampered (Tanner etal. 1995, review).

Problems of developing anti-parasite vaccines are abundant. Parasiteshave complex life cycles and each stage expresses different sets ofantigens. Moreover, the different stages are often associated withdifferent sites in the body. For most parasites little is known aboutthe immune mechanisms involved in natural immunity and about the stageof the parasite inducing this immunity.

Most often, no reproducible animal model is available to study thesemechanisms, thereby blocking a new approach in vaccine development. Asmentioned above, most available vaccines are based on attenuated liveparasites. These vaccines can sometimes be successful because the“vaccine parasites” follow the correct route of infection and deliver awide array of stage-specific antigens. However, such vaccines mustchallenge the acceptance of the public (e.g., Ancylostoma caninumvaccine), especially when they are for human use (e.g., Schistosomamansoni vaccine, Taylor et al. 1986). Moreover, live vaccines, ingeneral, have a short shelf-life and are relatively expensive. From thisperspective there is an obvious need for vaccines that are based on(recombinant) proteins derived from the parasite. However, theidentification of such protective proteins meets a great number ofdifficulties, as shown below as an example for Fasciola hepatica.

The trematode parasite Fasciola hepatica mainly infects cattle andsheep. Sometimes also humans get infected. The parasite causesconsiderable economic losses in, for example, western Europe, Australiaand South America. The metacercariae of Fasciola hepatica enter its hostby the oral route, penetrate the gut wall within four to seven hours(Dawes 1963; Burden et al. 1981; Burden et al. 1983; Kawano et al. 1992)and migrate through the peritoneal cavity towards the target organ, theliver. Oral infection of cattle results in almost complete protectionagainst a challenge, whereas sheep often die from an infection and donot acquire natural immunity. Both the natural host (cattle) and theanimal model (rat) acquire natural immunity after infection (Doy andHughes 1984; Hayes, Bailer and Mitrovic 1973). Therefore, rats are oftenused to study resistance in cattle. In the rat a large part of naturalimmunity is expressed in the gut mucosa, the porte d'entree of theparasite. In immune rats, about 80% of the challenge newly excystedjuvenile stages (NEJs) is eliminated in the route from the gut lumen tothe peritoneal cavity (Hayes and Mitrovic 1977; Rajasekariah and Howell1977; Doy, Hughes and Harness 1978/1981; Doy and Hughes 1982; Burden etal. 1981/1983). Based on natural immunity, a vaccine based on irradiatedFasciola gigantica metacercariae was developed for cattle (Bitakaramire1973). In the seventies and eighties many vaccination experiments havebeen performed with antigen extracts of adult and juvenile flukes(Haroun and Hillyer 1980, review). However, these studies lead toconflicting or disputable results. For example, subcutaneous orintramuscular injection of rats with adult or juvenile fluke extractsdid not result in protection (Oldham and Hughes 1982; Burden et al.1982; Oldham 1983). Adult fluke extracts given intraperitoneally inFreund complete adjuvant (FCA) or incomplete Freund adjuvant (IFA)resulted in about 50% protection (Oldham and Hughes 1982; Oldham 1983).Using very high antigen doses of Bordetella pertussis as additionaladjuvant this protection reached 80% to 86% (Oldham and Hughes 1982;Oldham 1983). Extracts of four-week-old juveniles givenintraperitoneally in AlOH₃ did not induce protection in the studies ofPfister et al. (1984/85), whereas 16-day old juvenile extracts provided86% protection in mice, without the use of adjuvant (Lang and Hall1977). Subcutaneous sensitization of cattle with sonicated 16-day-oldjuveniles resulted in more than 90% protection (Hall and Lang 1978).Intramuscular injection of calves with an isolated fraction from adultFasciola hepatica (Fh_(SmIII)), with an immunogenic 12 kD protein asmajor component, resulted in 55% protection (Hillyer et al. 1987).

Since 1990, several Fasciola hepatica vaccine candidate antigens havebeen isolated and/or produced. Most of these antigens are derived fromadult flukes and share homology with Schistosoma mansoni antigens.Glutathion S-transferases (GST) are enzymes amongst others active in thecellular detoxification system. Immunization of sheep (n=9) with GSTpurified from adult Fasciola hepatica, injected s.c. in FCA, with aboost immunization 4 weeks later in IFA, resulted in 57% protection(Sexton et al. 1990). Immunization of rats with GST provided noprotection (Howell et al. 1988). Vaccination trials in cattle performedby Ciba Animal Health Research (Switzerland) and The Victorian Instituteof Animal Science (Australia), resulted in 49% to 69% protection(Morrison et al. 1996).

Intradermal/subcutaneous immunization with recombinant S. mansoni fattyacid-binding protein Sm14 in FCA, provided complete protection againstFasciola hepatica challenge in mice (Tendler et al. 1996). PCTInternational Patent Publication WO 94/09142 suggests the use ofproteases having cathepsin L type activity, derived of Fasciolahepatica, in the formulation of vaccines for combating helminthparasites; immunization of rabbits with the purified mature enzymeresulted in rabbit antibodies capable of decreasing the activity of theenzyme in vitro.

However, levels of protection obtained with F. hepatica cathepsin L orhemoglobin in cattle were only 53.7% or 43.5%, respectively (Dalton etal. 1996). Cathepsin L belongs to a family of cysteine proteinases,secreted by all stages of the developing parasite. Cathepsin L from F.hepatica is most active at slightly acid or neutral pH (Dalton andHeffernan, 1989). The functions of this proteinase include disruption ofhost immune function by cleaving host immunoglobulin in a papain-likemanner (Smith et al. 1993) and preventing antibody mediated attachmentof immune effector cells to the parasite (Carmona et al. 1993).Moreover, cathepsin L is capable of degradation of extracellular matrixand basement membrane components (Berasain et al. 1997), and preparesmucosal surface to be penetrated by a parasite indicating that cathepsinL is involved in tissue invasion. Because of its crucial biologicalfunctions, cathepsin L proteases are considered important candidates forthe development of an anti-parasite vaccine.

Cathepsin L is synthesized as a preproprotein with a 15-amino-acid(“aa”)-long peptide presequence, a 91-aa-long peptide prosequence orproregion and a 220-aa-long poly)peptide enzymatic part. Of cysteineproteinases the preregion is removed immediately after synthesis and theproprotein comprising the proregion and the part that (constitutes themature enzyme) is transported to the Golgi. Conversion to the matureenzyme and thus conversion to an enzymatically active state, occurs inthe lysosomes and could be due to cathepsin D or to autoactivation. Insome cases precursors containing the proregion are secreted (North etal. 1990). Cathepsin L itself has a high affinity for a substrate withArg at the P1 position and a hydrophobic residue (Phe) at the P2position (Dowd et al. 1994). It also has autocatalytic activity andcleaves off its prosequence before it obtains its mature enzymaticactivity. Cathepsin L2 also cleaves peptides containing Pro at the P2position, and is therefore capable of cleaving fibrinogen and producinga fibrin clot.

Other potential candidates for an anti-fluke vaccine are hemoglobin,isolated from mature Fasciola hepatica (McGonigle and Dalton 1995) andcathepsin L secreted by adult Fasciola hepatica (Smith et al. 1993;Smith et al. 1994; Spithill 1995). Up to now, next to the irradiatedFasciola gigantica metacercariae (Bitakarami 1973) several antigens havebeen named as potential protein vaccines:

-   -   Fasciola hepatica hemoprotein    -   Fatty acid-binding protein Sm14 from Schistosoma mansoni    -   Thiol proteases with Cathepsin L-type activity    -   Glutathion S-transferase extracted from adult Fasciola hepatica    -   polypeptide from Fasciola species (Gln-Xaa-Cys-Trp-Xaa)    -   Serin proteases with dipeptidyl peptidase activity

However, none of these potential candidates have emerged as an effectivevaccine against Fasciola hepatica infection, and a large number ofquestions, such as: at what site in the host is immunity expressed?;against which stage of the parasite is immunity directed?; at which sitein the host this immunity is induced?; which immune mechanisms areinvolved in protection?; which stage of Fasciola hepatica inducesprotective immunity?; and, last but not least, which antigens induceprotection?, need to be answered before a successful vaccine can bedeveloped. It is clear that answering these questions is greatlyhampered by the lack of a suitable animal- or challenge model by whichparasitic infections can be studied. And even when animal models areavailable progress can only be slow because of the fact that theparasitic infection in the host under study takes a considerable time todevelop while its outcome depends on various factors that relate to thein time changing host-parasite relationship. For instance, although muchfocus has been directed to proteins, such as proteases, derived fromnewly excysted juvenile (NEJ) stages of Fasciola hepatica as candidateprotective antigens (see, for instance, Tkalcevic et al. 1995), no clearcut identification of truly protective proteins has been foreseen. Tothe contrary, early developmental stages of Fasciola hepatica displayrapid changes in protein and antigen expression during the early stagesof infection, and such changes may even assist the parasite to evade thehost immune response (Tkalcevic et al., Parasite Immunology 18:139-147,1996). It has, for instance, been demonstrated that in parasites,proteases are involved in the invasion of host tissues, the evasion ofimmune attack mechanisms and help provide nutrients for parasitesurvival.

Thus, both the abundance of possible different proteins or antigens thatneed to be studied and the lack of suitable challenge models hamper thepossible progress that is needed in the development of parasitevaccines. Crucial for progress in parasite vaccines are new methods tomeasure protective immunity in order to be able to study a variety ofcandidate protective antigens and to identify new candidate protectiveantigens. Thus, new animal models are needed that will increase thenumber of candidate proteins or substances that can be tested in time.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a very rapid method to study, investigateand evaluate natural immunity against a parasite under study. Theinvention provides an ex vivo animal or challenge model method torapidly study protective immunity directed against parasites andvaccines directed against parasitic infections. Ex vivo models are ingeneral designed to study organs or organ systems of animals, underanesthesia, out of the context provided by the natural body, but stillwithin the context of proper blood supply or the like. These models havein general a short execution time and provide less prolonged sufferingto the experimental animal than seen with in vivo models.

The invention provides an ex vivo gut model in the rat, or in othersmall experimental animals such as mice or chickens, or in other animalspecies. Challenge parasites are injected in one or more ex vivosegments of the intestines of the selected animal and parasites, such asNEJs, that then penetrate the intestinal wall are recovered in acontainer that holds the particular gut segment. In particular, segmentsof the small intestine, such as duodenum, jejunum or ileum can be used,however, segments of other parts of the intestine, such as stomach,colon, cecum or rectum can also be used depending on the selected routeof infection of the parasite under study. This model provided by theinvention is capable of measuring expression of resistance in the entireintestine by comparing segments that have been subjected to differentloads of parasites or to different stages of parasites. In addition, allthe trajects in the migration route of the parasite such as can be foundin gut mucosa, peritoneal cavity and liver and others, which areessential for the induction of mucosal resistance can be investigated.Such studies that are enabled by the invention provide knowledge aboutthe most efficient vaccination route and about possibilities for an oralvaccine. Another advantage of the ex vivo challenge model using ligatedgut segments is that migration of the pathogen from the gut lumen to theperitoneal cavity is limited to a small area, allowing the localizationand characterization of the protective immune response against theparasite in the gut mucosa. Moreover, the level of resistance induced bya previous infection or vaccination can be correlated with immunemechanisms against the parasite (in the experimental part demonstratedwith Fasciola hepatica) because the challenge infection does not settleand does not induce additional immune responses that interfere withthose that need to be studied. Especially the immunity and protectivemechanisms directed against those pathogens that penetrate mucosal orskin surfaces to infect the host, such as Fasciola hepatica, Paragonimuswestermani, Schistosoma mansoni, Toxocara canis, Dictyocaulus viviparus,Trichinella spiralis, Nematodiris spp, Nippostrongylus brasiliensis,Ascaris suum, Anisakis and other pathogens varying from prions toprotozoa, whether they may fully or partly penetrate the surfaces, canbe measured specifically well by the model provided by the invention.Parasites or other pathogens that fully penetrate the mucosal surfacesof the gut segments employed in the model can be recovered as shownabove, those that only partly penetrate the mucosal surfaces can berecovered from the blood or lymph vessels servicing the particularsegment.

Measuring the immunity and protective mechanisms directed againstparasites offers the possibility to modulate the effector phase of theimmune response in the host which will result in the development ofefficient vaccination strategies. In other words, the invention measuresthe capacity of proteins to be protective antigens for use as vaccineagainst infections. Because protection data are obtained the same daythe ex vivo model provided by the invention enables quick testing ofdifferent stages of many candidate vaccine antigens (protective proteinsor fragments derived thereof) for their capacity to induce resistanceand immunity.

One such candidate vaccine antigen provided by the invention is aprotective protein, or antigenic fragment derived thereof, the proteinat least comprising an amino acid sequence derived of a proregion of anenzyme. Several proteases are involved when a parasite penetrates amucosal or skin surface. Examples are serine protease, dipeptidylpeptidase-like protease, cysteine protease, proteases withcathepsin-like activity, but also enzymes like glutathion S-transferaseand many others are involved during the phase when the parasite ispenetrating a mucosal or skin surface. Surprisingly, the inventionprovides protective protein (fragments) derived of a proprotein of suchan enzyme or protease which elicits a better immune response than when amature enzyme is used. Optionally, it is possible to combine the immuneresponse directed against the proprotein with the immune responsedirected against the mature enzyme.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1: Mean recovery (±SD) of F. hepatica in the liver three weeksafter oral challenge of (a) non-immunized rats, (b) orally infectedrats; rats vaccinated with (c) MCF03 (d) MCF03+specol, (e)MCF03-KLH+specol, (f) MCF06 (g) MCF06+specoll (h) MCF06-KLH+specol (i)MCF04 (j) MCF04+specol (k) MCF04-KLH+specol.

FIG. 2: Nucleic acid sequences (2A) and deduced amino acid sequences(2B) of amplified cathepsin L proregions from different stages of F.hepatica, mRNA was isolated from adult flukes and newly excystedjuveniles, respectively, and converted to cDNA. With primer set TGG CATCAG TGG AAG CGA ATG // ATA ACC AGA TTC ACG CCA GTC (SEQ ID NOS: 7, 8),cathepsin L was amplified using adult F. hepatica cDNA as template(da13pro). With primer set TGG CAY GAR TGG AAR MGN ATG // RTA NCC RTAYTC NCK CCA RTC (SEQ ID NOS: 9, 10), the proregion of cathepsin L wasamplified, using cDNA from newly excysted juveniles as template (da210pro, da211pro). Amplified products were cloned into a pCR™ vector andsequenced. Nucleic acid sequences (Wijffels (SEQ ID NO: 19), da13pro(SEQ ID NO:21). da210pro (SEQ ID NO:23), and da211pro (SEQ ID NO:25) anddeduced amino acid sequences of cathepsin L proregions obtained(Wijffels (SEQ ID NO: 20), da13pro (SEQ ID NO:22), da210pro (SEQ IDNO:24), and da211pro (SEQ ID NO:26) were aligned with the sequence fromWijffels et al. (1994).

FIG. 3: Alignment of cathepsin L proregions from Fasciola hepatica (SEQID NO: 27) (F-hep, Wijffels et al. 1994), Schistosoma manosoni (5-man1(SEQ ID NO: 28), Michel, Klinkert and Kunz 1994; S-man2 (SEQ ID NO: 29),Smith et al. 1994), Schistosoma japonicum (SEQ ID NO: 30) (S-jap, Dayand Brindley 1995) and Homo sapiens (SEQ ID NO: 31) (H-sap, Joseph etal. 1988). Amino acid residues that match F. hepatica sequence exactlyare indicated by a box.

The proregion of F hepatica cathepsin L showed 41.8%, 38.5%, 30.8% and20.2% homology with the cathepsin L proregion of S. mansoni (1), S.Japonicum, H. sapiens and S. mansoni (2), respectively.

FIG. 4: Schematic representation of synthetic peptides derived fromcathepsin L, and their reactivity with sera from F. hepatica infectedcalves, tested by ELISA. Five synthetic peptides of cathepsin L wereproduced, according to the amino acid sequence from Wijffels et al.(1994), representing possible immunogenic regions on the cathepsin Lmolecule (based on the antigenicity index from Jameson and Wolf).Peptides and purified cathepsin L, respectively, were coated onto ELISAplates and tested with sera ( 1/50 dilution) from two calves, sampled atregular intervals after infection with F. hepatica. Strong reactivity(OD value>0.8) of both calves was detected with both peptides MCF05 andMCF04 and with purified cathepsin L, from 54 days after infection.

FIG. 5: Sensitivity (A) and specificity (B) of F. hepatica peptide MCF04ELISA.

A) Peptide MCF04, derived from cathepsin L (FIG. 4), was coated ontoELISA plates and tested with sera from five sheep, sampled at regularintervals after infection with F. hepatica. From week 5 after infection,OD-values increased and remained high until at least week 16 afterinfection.

B) Peptide MCF04 was coated onto ELISA plates and tested with sera fromsheep, infected with Fasciola hepatica (n=5), Echinococcus granulosus(n=9), Nematodirus battus (n=3) Haemonchus contortus (n=12), Toxoplasmagondii (n=12), Eimeria spp. (n=12), Ostertagia circumcincta (n=8),Cooperia oncophora (n=12), Taenia ovis (n=8), and with parasite-freesheep (n=12).

DETAILED DESCRIPTION OF THE INVENTION

In a preferred embodiment of the invention, the invention providesprotective protein(fragments) derived of a proregion of such an enzyme,preferably a protease. In yet another embodiment, the immune responsedirected against the proregion is combined with the immune responsedirected against the mature enzyme.

One such candidate vaccine antigen provided by the invention is aprotective protein, or antigenic fragments thereof, derived from NEJs ofFasciola hepatica. The protective status of rats vaccinated withcandidate vaccine antigens, such as can be prepared from NEJ proteins,can be measured via vaccination studies using the ex vivo model providedby the invention, for example, by measuring the protective status ofrats previously immunized with a selected protein. Various proteinsderived from NEJs of Fasciola hepatica and isolated, for instance viaSDS-PAGE gel electrophoresis and electroblotting, or via exclusion bymolecular size, or filtration, and further identified by apparentmolecular weight and by N-terminal sequencing can be studied. Theinvention, as an example, provides among others a protective protein orfragments thereof corresponding to an immunodominant protein found withNEJs with an apparent molecular weight of 30 to 32 kD and an N-terminalamino acid sequence of XXDVSWPFWDRMYNY (SEQ. ID NO:1) (amino acids arelisted in the one-letter code, x=unknown amino acid). Also provided bythe invention are nucleotide sequences encoding protective proteins or(poly) peptides provided by the invention.

A preferred embodiment of the invention is a protective protein, orfragments thereof, which protein is at least comprising an amino acidsequence derived of a proregion or prosequence of a protease, forexample, a protease which is (at least partly) encoded by a nucleic acidhaving a nucleotide sequence corresponding to a nucleotide sequence asshown in FIG. 2.

Methods to derive such sequences from (partly) isolated or identified(parasite)proteins are known in the art, for example, it is possible toidentify immunogenic determinants or fragments by studying theantigenicity index by, for example, computer analysis. Furthermore,nucleotide sequences encoding the enzymes or proteases are known in theart. Sequences encoding cathepsin-like proteases are, for example, shownby Wijffels et al. (1994). Often, a part of a sequence encoding a matureenzyme is known, which enables an average skilled artisan to identifycorresponding nucleic acid sequences encoding corresponding pre- and/orproregions. Such nucleic acid and/or protein sequences can be obtainedfrom both adult or juvenile stages of an organism. A preferredembodiment of the invention provides a protective protein, or fragmentsthereof, corresponding to a proregion derived of a protease mainly foundin a juvenile stage of a parasite, preferably the parasite being aFasciola species. By using isolated nucleotide sequences inamplification or screening methods, varying or different parasiticsequences that encode functionally equivalent proteins or (poly)peptides can be identified in and isolated from related parasites. Allsuch nucleotide sequences or fragments thereof can be molecularly clonedby methods known in the art in suitable expression systems to generaterecombinant proteins that can be used in anti-parasite vaccines or fordiagnostic purposes, as described above.

An example of an immunogenic determinant or fragment or fragments asprovided by the invention is a fragment derived of a proregion of aprotease. A typical example is a peptide corresponding or related with apeptide, such as MCF03 or MCF06, or a peptide found at an overlappingposition, in a proregion of a protease, examples can also be found inFIGS. 2 and 3. Other corresponding fragments or peptides can be found inrelated (proregions of) proteases. In the experimental part suchpeptides that are derived of a proregion of a cathepsin-like proteaseare described. It is well known in the art that synthetic peptides canbe rendered more immunogenic by replacing amino acids with others. Alsodeletion or insertion of (an) amino acid(s) in such peptides ispracticed. Guidance can be found by using techniques such as PEPSCAN, orreplacement-net mapping, in this way more immunogenic peptides arederived from original peptide sequences. Immunogenicity can further beincreased by replacing L-amino acids by D-amino acids. In a preferredembodiment of the invention, such vaccines comprise at least a proteinor (peptide) fragment thereof derived from a proregion of cathepsin-likeproteases (such as Cathepsin B, H, L, S), for example, derived from S.Mansoni, Tryponasoma Cruzei or T. Congolense, or vertebrate cathepsinderived, for example, from chicken, rat or human lives, or othercathepsin-like proteases. Cleavage sites, identifying the pre- andproregions of such proteases can easily be found by comparing sequencecharacteristics and, for example, by following Von Heijne's rule.Assessment or measuring of the protective value or capacity of suchproteins or vaccines can of course be performed in the ex-vivo modelalso provided by the invention. The nucleotide sequences alone, orincorporated in suitable vector systems or constructs can also beemployed in DNA vaccination protocols. Such sequences can for instancebe derived by amplification techniques, such as PCR, using degenerateprimers deduced from (partly) known amino acid sequences correspondingto protective proteins provided by the invention. Amplified nucleotidefragments can be cloned and sequenced via standard techniques and soprovide the isolated nucleotide sequence of genes or fragments thereofencoding the protective proteins or (poly) peptides provided by theinvention. Such proteins or fragments can, in isolated and/orrecombinant form, be used as vaccine antigens, alone or in combinationwith other preparations serving as vaccine or can be used as diagnosticantigen in diagnostic tests. Also, antibodies, be they polyclonal ormonoclonal or synthetic antibodies or antibody fragments specificallydirected against or prepared against protective proteins or (poly)peptides provided by the invention are part of the invention.

Furthermore, a diagnostic test comprising the protective protein or anantibody directed against the protein or a nucleotide encoding theprotein are also part of the invention. Furthermore, a diagnostic testwhich measures proteins excluding the protein, or antibodies againstproteins, excluding antibody directed against the protein, and whereinthe diagnostic test is specifically designed to be used as anaccompanying test to the use of a vaccine which specifically includesthe protein is also part of the invention. With such an accompanyingtest infected animals can be differentiated from vaccinated animals. Anexample of such a diagnostic test is given in the experimental part inthis description. Herein it is shown that antibodies directed against anprotective epitope derived from a proregion can be differentiated fromantibodies directed against the mature part of the enzyme, allowing thedifferentiation of infected animals from vaccinated animals. It is ofcourse preferred that the animals are vaccinated with a vaccinecomprising a protein (fragment) derived of a proregion of an enzyme,such as a protease, as provided by the invention. Such differentiationis not possible when animals are vaccinated with a (mature) proteasewith enzymatic activity.

In a preferred embodiment of the invention, a vaccine comprises mainly aprotein (fragment) derived of a proregion of a protease, where as adiagnostic test comprises mainly a protein (fragment) derived of amature enzyme part of the protease, whereby combining such a vaccinewith such a test allows controlled eradication of a parasite infection.

In addition, the invention provides a diagnostic test measuring anantibody directed against an immunodominant, species specific, epitopeon a cathepsin-like protease, preferably wherein, the species isFasciola hepatica. The test, comprising, for example, a peptidecorresponding to peptide MCF04, or a peptide related thereto, allows,for example, biological differentiation of animals infected withFasciola hepatica from animals injected with other parasites, such as D.viviparus, which otherwise have a strong immunoreactivity withCathepsin-L protease as a whole.

As described herein above, and further described in the experimentalpart of this description, without limiting the invention thereto, theinvention among others provides a protective protein, or antigenicfragment derived thereof, and related nucleic acid sequences, that areat least comprising and/or encoding an amino acid sequence derived of orproregion of an enzyme, such as a protease, for example, for inclusionin a vaccine, for example, in parasitic infections. The invention alsoprovides use of such a vaccine in animals, preferably mammals. Vaccinecandidates are, for example, vaccines for protection against parasiticinfection in ruminants, such as those susceptible to Fasciolainfections, or in humans, such as those susceptible to Schistosomainfections.

EXAMPLES Experimental Part

Materials and Methods

Rats

Specific pathogen free female Wistar rats (Charles River, Sulzfeld) wereselected for all experiments. Rats were provided with food and water adlibitum. Rats were food deprived during 16 hours before primary andchallenge infection. Rats were six weeks of age at the time of primaryinfection or first vaccination. Rats were ten weeks of age at the timeof the challenge infection, with the exception of rats used to study theduration of resistance. These rats were 19 weeks old at the time ofchallenge infection.

Fasciola hepatica

Fasciola hepatica metacercariae were produced within the ID-DLOinstitute. In vitro excystment of metacercariae was performed by themethod of Smith and Clegg (1981). NEJs were counted under a microscope(magnification 160×) directly after excystment. NEJs were kept in 300 μlof RPMI-1640 culture medium (ICN-Biomedicals BV, Zoetermeer, Holland) at37° C. until use (less than one hour after excystment).

Primary Infection

Twenty-five Fasciola hepatica metacercariae were orally administered torats in 1 ml of tap water. After delivery of the pathogen syringe andcannula were flushed to check delivery of the metacercariae.Metacercariae that stayed behind were administered in another ml of tapwater.

Expression of Resistance

Total resistance: quantification of the number of challenge parasitesreaching the target organ, the liver.

To measure the total level of protection against Fasciola hepatica ratswere orally challenged with exactly 200 metacercariae. After delivery ofthe pathogen syringe and cannula were flushed to check delivery of themetacercariae. Metacercariae that stayed behind were administered inanother ml of tap water. Three weeks after challenge, infection ratswere killed, livers removed and placed in separate petri-dishescontaining 50 ml of RPMI-1640 culture medium. Livers were incubated at37° C. Every hour (up to six hours), livers were cut into smaller piecesand placed in new petri-dishes. NEJs recovered were counted.

Resistance at Gut Level: Quantification of the Number of NEJsPenetrating the Gut Wall, Using an Ex Vivo Infection Model

Rats were anaesthetized by ether inhalation and immediately thereafterinjected with 50 mg/kg of nembutal (Compagnie Rousselot, Paris, France)intraperitoneally and 0.05 mg/kg of atropin (AUV, Cuyk, Holland)subcutaneously. During the experimental procedure, additional nembutal(16 mg/kg) was injected subcutaneously three hours after etherinhalation. Forty-five minutes after anesthetization, an incision (1.5cm) was made below the diaphragm and a loop of the small intestine ofabout 7 cm in length was taken out of the body cavity. A segment orsegments of about 5 cm was delimited with two linen threads (B. Braun,Melsungen AG), at standard locations from the stomach. To studyresistance at different locations in the intestine, segments of theduodenum (1 to 5 cm from the stomach; n=6), the mid jejunum (40 to 60 cmfrom the stomach; n=6) and the ileum (70 to 90 cm from the stomach; n=6)were prepared. In the segment or segments NEJs were injected accordingto the method of Burden et al. (1983). After injection, needle andsyringe were flushed three times with 1 ml of medium in a petri dish.NEJs that remained behind in syringe and/or needle during inoculationwere quantified under a microscope (rest fraction), and the infectiondose was calculated (counted dose minus rest fraction).

During the experiment, the gut loop or loops including the segment orsegments was or were kept outside the body cavity and the incision wasclosed with one or two surgical staples. Per experiment, eight rats werelaid onto perspex plates, the gut loops were led through holes in theplates and hung freely in 50 ml beakers well below the surface ofRPMI-1640 medium. The beakers with 50 ml medium were changed every hourand NEJs that had migrated through the gut wall into the beaker werequantified by light microscopy (peritoneal fraction; magnification100×). During the experiment, the whole system was kept at bodytemperature: 1) by placing the beakers in a water bath of 37° C., 2) bywarming the rats pumping warm water from a central heater below theperspex plates on which the rats were laying and 3) by warming the ratsusing an infrared lamp, when necessary as indicated by measurement ofthe body temperature. The rats were killed after six hours, gut segmentsremoved and segment size and distance to the stomach determined. Thelumina of the segments were flushed with medium and NEJs remaining inthe gut lumen were quantified by light microscopy (luminal fraction).The segments were finally fixed according to the “Swiss roll” method(Bexter, 1982) in methylbutane (−150° C.) and stored at −70° C. forimmunohistochemistry.

Reproducibility of the Ex Vivo Gut Model

To determine the number of NEJs left in the gut wall (gut fraction)after the experiment, each gut segment was cut into 10 μm frozensections. Every fifth section was collected to score any NEJ (sizeNEJ±100 μm), air-dried and fixed for ten minutes in acetone (Merck).Fixation and all subsequent washings and incubations were performed atroom temperature. After fixation peroxidase activity in the gut wall wasblocked: sections were incubated for 20 minutes in 0.1 M Tris-HCl pH7.5, containing 2% NaN₃ and 0.2% H₂O₂. Sections were then washed forfive minutes in three changes of Tris-buffered saline pH 7.4 (TBS),stained for five minutes in 0.1 M Tris-HCl pH 7.5, containing 1 mg/ml3,3′-diaminobenzidine (Sigma, St. Louis, USA) and 0.015% H₂O₂, washedfor five minutes in three changes of phosphate-buffered saline pH 7.6(PBS) and incubated for one hour in PBS containing 2% normal rat serum(NRS) and 4% bovine immune serum. This serum was raised in afive-month-old calf by two oral infections with 4500 and 2250 Fasciolahepatica metacercariae, with an interval of 11 weeks. Antiserum wasobtained eight weeks after the second infection. After three washingswith PBS, sections were incubated for one hour withperoxidase-conjugated rabbit anti-cow immunoglobulin (Dakopatts,Glostrup, Denmark), diluted 1:500 in 2% NRS in PBS. Subsequent washingwas performed and peroxidase activity was visualized by an eight-minuteincubation in a freshly made, filtered solution of 0.05 M NaAc pH 4.4,containing 0.2 mg/ml 3-amino-9-ethylcarbazole (Sigma, St. Louis, Mo.,USA) and 0.015% H₂O₂. After staining, the sections were washed inrunning tap water and mounted in aquamount (BDH Laboratory supplies,Poole, England). Microscopically counting of NEJs was performed andsuccessive sections were compared to prevent scoring NEJs twice.

Induction of Resistance

Gut Level

1) Five rats were orally infected with 25 metacercariae and treated fourhours later with the flukicide triclabendazole (100 mg/kg, Fasinex,CIBA-GEIGY, Basel, Switzerland). Flukicide treatment was repeated thefollowing three days. After five weeks expression of resistance at gutlevel was measured, using the ex vivo gut model, and “breakthrough”infections in the liver were investigated at autopsy. To confirm thatFasinex treatment did not influence migration of the challenge parasitesthrough the gut wall, non-infected, fasinex-treated rats were used aschallenge controls.

2) Four rats were primed with 18 to 25 NEJs directly in the jejunum.During four hours, NEJs penetrating the gut wall were captured using theex vivo gut model. During primary infection, rats were anesthetized byan intraperitoneal injection with ketamine (40 to 60 mg/kg; Alfasan,Woerden, NL), and a subcutaneous injection with xylazine (3 to 8 mg/kg;Rompun, Bayer, Germany) and atropine (0.05 mg/kg). One day before andone day after infection, rats were treated with the antibiotic duoprim(0.5 ml/kg, subcutaneously; Pitman-Moor, Houten, NL). The sedativefiadyne (1 mg/kg, intramuscularly; Schering-Plough, Amstelveen, NL) wasgiven the first three days after infection. After four weeks expressionof resistance at gut level was measured and “breakthrough” infections inthe livers were investigated at autopsy.

Peritoneal Cavity/Liver

NEJs of the primary infection were injected in the peritoneal cavity(n=3, 13 to 17 NEJs) or between the liver lobes (n=8, 7 to 25 NEJs). Forliver infection, a small incision (1 cm) was made below the diaphragm.NEJs in 100 μl of RPMI-1640 were injected between the liver lobes.During the operative procedure rats were anaesthetized as describedabove. For intraperitoneal infection rats were anesthetized by etherinhalation and immediately thereafter intraperitoneally injected withthe NEJs.

Preparation of Fasciola Hepatica Antigen Extracts

After in vitro excystment, NEJs were washed with PBS. Three hundred mgof NEJs in 3 ml of PBS were sonificated (Sonicor UPP-400, SonicorInstrument Corporation-copaque, NV) five times for 30 seconds at 20 kHzon ice. The suspension was extracted over night at 4° C. and thereaftersonificated again. The extract was centrifugated for 20 minutes at10,000 g and the supernatant stored in aliquots of 1 ml at −70° C.Concentration of protein in the extract was 3 mg/ml, as determined by aBradford assay.

Adult Fasciola hepatica were obtained from the livers of cattle andthoroughly washed with HMEM-medium and subsequently with PBS at 4° C.Flukes were ground using a Sorvall omnimixer (model 17106) ten times for30 seconds on ice. The subsequent sonification and extraction procedureswere performed as described above.

YM-30 Filtration NEJ-Antigen

Freshly prepared NEJ extract (10 ml of a 3 mg/ml extract) was diluted inPBS to a volume of 30 ml and filtrated through a YM-30 membrane (Amicon,62 mm) at 1 Bar, at 16° C. (Amicon model 8200). The 5 ml rest fractionwas replenished with 5 ml of PBS and filtrated again to a 5 ml restfraction. This procedure was repeated two times. Finally, the 40 mlfiltrate (25 μg/ml) was stored at −70° C. in aliquots of 1 ml. Otherfiltrates, containing more protein, i.e., 183 μg/ml were prepared andstored likewise.

Vaccination Regimes

Rats were primed with 100 μg of NEJ or adult stage Fasciola hepaticaantigen intraperitoneally. After three weeks, an intraperitoneal boostimmunization with 500 μg of antigen was given. One week after the boost,immunization resistance against a challenge infection was determined. Tomeasure the total level of protection rats were orally challenged with200 metacercariae and to measure the level of protection expressed atgut level rats were intrajejunally challenged with NEJs (for recoveryprocedures see “expression of resistance”).

Doses of the YM-30 filtrate used were 20 μg and 65 μg for primary andboost immunization, respectively.

SDS-PAGE and Western Blotting

Sodium dodecyl sulfate polyacrylamide gel electroforesis (SDS-PAGE) wasperformed using the Tris-Tricine buffer system (Schagger and von Jagow,1987) with 10% to 20% (w/v) polyacrylamide gradient gels or 15% slabgels (8 by 10 cm). 12.5 μg of protein was applied per gel in thepresence of β-mercaptoethanol. To determine the molecular weights of theNEJ proteins a prestained MW marker from BRL (Bethesda ResearchLaboratories, Breda, The Netherlands) was added to the gel (MW range:14.3-200 kD). After electrophoresis at 20 mA for 3.5 hours, separatedproteins were electrophoretically transferred (16 hours, 20 mA, RT) ontoa polyvinylidene difluoride (PVDF)-type membrane (Applied Biosystems,Inc) using a buffer system, containing 10 mM3-cyclohexylamino-1-propane-sulfonic acid (CAPS) pH 11 (Aldrich) in 10%methanol.

N-Terminal Sequencing

Blotted proteins were visualized by staining with 0.1% CoomassieBrilliant Blue R-250 (Sigma). The regions staining with CBB or theprotein band staining in immunoblotting with the sera were excised fromthe PVDF-membrane and 2 cm membrane was subjected to Edman degradationsequencing using an Applied Biosystems Protein Sequencing system (model476A). Analysis was performed at “The Centre for Biomembrane and LipidEnzymology, Department of Biochemistry, University of Utrecht.”

Immunostaining

Four immunostaining 4 mm PVDF-strips were saturated for one hour with10% normal rabbit serum (NRS) in PBS-0.5 M NaCl-0.05% TWEEN®-80, pH 7.2(PBS-NT). Subsequently strips were incubated for 16 hours with 40 μl ratserum or 40 μl calve serum in 2 ml of PBS-NT containing 2% NRS. The calfsera were obtained from five-month-old calves, 12 weeks after oralinfection with 4500 Fasciola hepatica metacercariae. After a three-hourincubation with 20 μg of mAb anti-rat IgG1 (culture supernatant, TNOLeiden, The Netherlands) and 16 μg of mAb anti-bovine IgG1 (van Zaane etal.) in 2 ml PBS-NT containing 2% NRS, HRPO-conjugated rabbit anti-mouseIg (Dakopatts), 1/500 diluted in PBS-NT containing 2% normal rat serumwas added for two hours. Chloronaphtol (Sigma; 0.5 mg/ml4-Chloro-1-naphtol and 0.015% H₂O₂ in Tris-buffered saline pH 7.4) wasused as substrate. One hour after application of the substrate stainingwas stopped by washing the strips with aqua dest.

All incubations were performed at room temperature and between allincubation steps strips were washed three times during ten minutes withPBS-NT.

Peptide Synthesis

Reagents

N-methylpyrrolidone (NMP), N,N-dimethylformamide (DMF),N-hydroxybenzotria-zole (HOBt),2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU) and piperidine were peptide synthesis grade and obtained fromPerkin Elmer/ABI (Warrington, UK). Acetonitrile was gradient grade,diisopropylethylamine (DIEA), trifluoroacetic acid (TFA), thioanisole(TA), phenol, and ethanedithiol (EDT) were synthesis grade and wereobtained from Merck (Darnstadt, Germany). Before use, diethyl ether waspurified over a column of activated basic aluminum oxide and DIEA wasdistilled twice over ninhydrin and potassium hydroxide. Fmoc-amino acidderivatives and Rink resin (4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy resin) were obtained from Saxon Biochemicals (Hannover,Germany).

Peptide Synthesis

Five synthetic peptides were produced of about 20 amino acids in length,according to the sequence of cathepsin L from Wijffels et al. (1994).The peptides were derived from possible immunogenic determinants on themolecule, based on the antigenicity index as described by Jameson andWolf. Peptides MCF03 and MCF06 were derived from the prosequence ofcathepsin L, peptides MCF05, MCF04 and MCL13 from the enzymatic part ofthe molecule. Peptide MCF03 included aa 15 to 33(Gly-Ser-Asn-Asp-Asp-Leu-Trp-His-Gln-Trp-Lys-Arg-Met-Tyr-Asn-Lys-Glu-Tyr-Asn)(SEQ ID NO:2), peptide MCF06 aa 25 to 42(Lys-Arg-Met-Tyr-Asn-Lys-Glu-Tyr-Asn-Gly-Ala-Asp-Asp-Gln-His-Arg-Arg-Asn)(SEQ ID NO:3), peptide MCF05 aa 103 to 122(Ala-Asn-Asn-Arg-Ala-Val-Pro-Asp-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu)(SEQ ID NO:4), peptide MCF04 aa 110 to 129(Asp-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val-Lys-Asp-Gln-Gly-Asn-Cys)(SEQ ID NO:5) and peptide MCL13 aa 296 to 311(Gly-Glu-Arg-Gly-Tyr-Ile-Arg-Met-Ala-Arg-Asn-Arg-Gly-Asn-Met-Cys) (SEQID NO:6). The molecular masses of the peptides were in accordance withthe expected values.

We used a Hamilton Microlab 2200 (Reno, Nev., USA) to synthesize up to40 peptides simultaneously at 30 mmol scale. The Hamilton Microlab 2200was programmed to deliver washing solvents and reagents to two rackswith 20 individual 4 ml columns with filter, containing resin forpeptide synthesis. The columns were drained automatically after eachstep by vacuum. The coupling cycle was based on Fmoc/HBTU chemistry(Fields et al. 1991) using double coupling steps of 40 minutes. PeptidesMCF03, MCF06 and MCF05 were synthesized with an additional cysteine atthe N-terminus. After coupling of the last amino acid, the Fmoc groupwas removed using 30% (v/v) piperidine/NMP for three and for 15 minutes.The peptides were washed with NMP (five times), acetylated usingNMP/acetic anhydride/DIEA (10/1/0.1; v/v/v) for 30 minutes, washedsuccessively with NMP and ethanol, and then dried. Peptides weredeprotected and cleaved in two hours using 1.5 ml of a mixture ofTFA/phenol/TA/water/EDT (10/0.75/0.5/0.5/0.25; v/w/v/v/v/) and thenprecipitated twice by adding hexane/diethylether (1/1; v/v). Theprecipitate was dried and lyophilized from water/acetonitrile (1/1;v/v).

HPLC and Mass-Spectrometry

For analytical HPLC we used two Waters pumps model 510, a Watersgradient controller model 680, a Waters WISP 712 autoinjector, and aWaters 991 photodiode array detector. A micromass Quattro II sq massspectrometer, coupled with the HPLC system, was used to determine themolecular masses of the individual peaks by electrospray ionization. Theproducts were analyzed in a linear gradient from 10% (v/v)acetonitrile/water with 0.1% (v/v) TFA to 70% (v/v) acetonitrile/waterwith 0.1% (v/v) TFA in 30 min on a Waters Delta Pak C18-100A (3.9×150mm, 5 mm) column at 1 ml/minute.

Conjugation of Peptides to Keyhole Limpet Haemocyanin (KLH)

Peptides were conjugated to KLH carrier protein, usingm-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). To 1 mg of KLH(Calbiochem, 10 mg/ml in 0.1 M phosphate buffered saline pH 7) 100 ml ofMBS (Pierce, 40 mg/ml in dimethylformamide (Merck)) and 300 ml ofacetonitrile were added in drops and the mixture was incubated for onehour on ice. Then 1.1 ml of PBS was added and the activated carrier wasseparated from excess MBS using a PD10 column (Pharmacia). One mg ofpeptide was added to 2.3 mg of activated KLH and incubated for one hourat room temperature. Then peptide and conjugate were separated bydialysis against PBS and the conjugate stored at −20° C.

Vaccination of Rats with Synthetic Peptides Derived from Cathepsin L

Rats (four per group) were vaccinated in the hind thigh muscles with 100mg of peptide (i) in PBS, (ii) mixed with specol (ID-DLO, Lelystad),according to the manufacturer's instructions, (iii) coupled to KLH andmixed with specol. After three weeks, an intraperitoneal boostimmunization was given with 100 mg of the corresponding peptide, withoutthe use of specol. One week after the boost immunization rats werechallenged orally and the parasite load in the liver was measured threeweeks later.

PCR, Subcloning and Sequencing

mRNA was isolated from 450 μl of packed NEJs (±45.000 NEJs) and twoadult F. hepatica, respectively, using a QuickPrep mRNA Purification Kit(Pharmacia Biotech).

cDNA was produced using a First-Strand cDNA Synthesis Kit (PharmaciaBiotech). The PCR amplification reactions were performed in 25 μlreaction volumes of PCR buffer II (perkin Elmer) containing 100 ng ofcDNA, 2.5 mM MgCl₂, 200 μM-dTNPs, 1.08-1.46 μM of F. hepatica specificprimers or 1 μM oligo (dT), and 0.5 unit of Taq DNA polymerase gold(Perkin Elmer). The sequences of the oligonucleotide primer sets, usedto amplify the specific cathepsin L sequences were the following:

Cathepsin L sequence primer set sequences (5′-3′) (i) prosequence TGGCAT CAG TGG AAG CGA ATG (fw) (SEQ ID NO:7) adult F. ATA ACC AGA TTC ACGCCA GTC (rv) (SEQ ID NO:8) hepatica (ii) proprotein TGG CAT CAG TGG AAGCGA ATG (fw) (SEQ ID NO:7) adult F. Oligo (dT) (rv) hepatica (iii)prosequence TGG CAY GAR TGG AAR MGN ATG (fw) (SEQ ID NO:9) NEJ RTA NCCRTA YTC NCK CCA RTC (rv) (SEQ ID NO:10) (iv) proprotein TGG CAY GAR TGGAAR MGN ATG (fw) (SEQ ID NO:9) NEJ Oligo (dT) (rv) (v) proprotein TGCCCN TTY TGG AAR MGN ATG (fw) (SEQ ID NO:11) NEJ Oligo (dT) (rv)

The amplification reactions were performed in a preheated Perkins ElmerCetus DNA Termal Cycler (80° C.), ten minutes at 92° C. followed by 30cycles of 30 seconds at 94° C., 30 seconds at 62° C. and two minutes at72° C.

Amplified fragments were inserted by TA cloning into the LacZ gene of apCR™ II vector, according to the manufacturer's instructions (TA CloningKit, Invitrogen). After transformation of TA Cloning One Shot competentcells, clones harboring inserts were distinguished by their white color.To verify size of the inserts, plasmid DNA was isolated using the WizardPlus SV Minipreps DNA Purification System (Promega) and digested byBamHI and EcoRV (Pharmacia LKB Biochemicals), according to themanufacturer's instructions.

Sequencing of the cloned material was done using the chain terminationreaction described by Sanger et al. (1977). Of each product, at leasttwo positive clones were sequenced, using both the M13 Reverse andForward primers. Using these primers, we were not able to sequence thewhole procathepsin L molecule at one go. Additional primers (5′-3′) weredesigned, based on the nucleotide sequences obtained, to sequence themissing parts:

NEJ: (SEQ ID NO:12) ATC AGG GAC AAT GGT TCC (fw; position 398-417) (SEQID NO:13) GAA GTG AGA TTG AGG ATC CAC (rv; position 752-772) (SEQ IDNO:14) CAA TAC AGG AAA GAG CTT GG (fw; position 638-657) adult: (SEQ IDNO:15) ACT GTG GTT CCT GTT GGG C (fw; position 407-425) (SEQ ID NO:16)GTG TGA ATA AAT ACC ACT CCT G (rv; position 779-800)Of these primers, the use for either forward (fw) or reversed (rv)sequencing is indicated, as is their position with respect to thecathepsin L sequence from Wijffels et al. (1994).Cathepsin L Peptide Enzyme Linked Immunosorbent Assay (ELISA) for theDiagnosis of F. hepatica Infection in Cattle and SheepExperimental Sera

To obtain mono-specific anti-Fasciola sera, 24 Holstein Frisian calvesof five to eight months of age, reared free of parasites, were infectedwith 100 to 3000 F. hepatica metacercariae. Serum samples were taken atweekly intervals. Calves were monitored for infection by weekly countingof the number of eggs in the feces. At slaughter, flukes were detectedin the bile duct of all calves. In addition, four- to eight-month-oldcalves were mono-infected with D. viviparus (Dv; n=4), Ostertagiaostertagi (n=1), Nematodirus helvetianus (n=1), Cooperia oncophora (n=1)or Ascaris suum (n=1). Serum samples were taken when all the infectedcattle shed parasite eggs or D. viviparus larvae. Detailed informationon these sera is provided elsewhere (de Leeuw et al. 1993).

Five ewes of the Texel Sheep Breed, between three and twelve months ofage, reared free of parasites, were infected with 20 F. hepaticametacercariae. Serum samples were taken at weekly intervals. Sheep weremonitored for infection by weekly counting of the number of eggs in thefeces. At slaughter, flukes were detected in the bile duct of all sheep.Monospecific sera against Haemonchus contortus (n=12) originated fromsheep infected repeatedly (five to 50 times) with doses of 5,000 to20,000 larvae. Antisera against Ostertagia circumcincta (n=8) originatedfrom sheep infected once with 30,000 larvae. Monospecific sera againstTaenia ovis (n=8) originated from sheep that had grazed on a pasturecontaminated with T. ovis eggs. Cysticerci were found in all sheep atslaughter. Monospecific sera against Cooperia oncophora (n=12)originated from sheep infected once with 20,000 larvae. Monospecificsera against Nematodirus battus (n=3) originated from sheep infectedfive times with 5,000 larvae. Blood samples were taken ten to 15 weeksafter infection, when all infected sheep shed parasite eggs or oocysts.Negative control sera were collected from parasite-free sheep (n=12).

Purification of Cathepsin L from Excretory/Secretory Products from F.hepatica

Adult flukes, collected from the bile ducts of experimentally infectedcows, were washed three to four times, for one hour, with 0.01M PBS (pH7.0). Twenty flukes were incubated per liter of HMEM medium containingstreptomycin (100 μg/ml) and penicillin (100 IU/ml) at 37° C. for sixdays. The medium was refreshed each day and the supernatants collectedfrom days 3 to 6 were pooled. This pool was centrifuged at 4° C. at10,000 g for one hour and the supernatant stored at −70° C. until use.The average protein concentration was 10 μg/ml. The total protein yieldwas 1 mg per 10 g of flukes.

These excretion/secretion antigens were filtered through a YM-30membrane (Amicon). The pH of the filtrate was adjusted to pH 9.5 with0.5 M Tris-HCl pH 11, and subjected to ion exchange chromatography on adyethylaminoethyl (DEAE)-Sephacel column (Pharmacia LKB, Uppsala,Sweden), equilibrated with 0.05 M sodiumcarbonate buffer, pH 9.5. Afterapplication of the filtrate, the DEAE-Sephacel column was washed with0.05 M sodiumcarbonate buffer, pH 9.5, containing 200 mM NaCl. Thecolumn was subsequently eluted with the same buffer containing 500 mMNaCl. The eluate was subjected to SDS-PAGE, electroblotting and CBBstaining and revealed one protein band. The 15 N-terminal amino acids,Ala-Val-Pro-Asp-Lys-Ile-Asp-Trp-Arg-Glu-Gln-Gly-Tyr-Val-Thr (SEQ IDNO:32), showed 95.4% homology to the cathepsin L sequence of Wijffels etal. (1994).

ELISA Procedure

ELISA plates (Greiner nr. 655001, Alphen aan de Rijn, The Netherlands)were coated with 100 μg of peptide MCF02, MCF03, MCF04, MCF05 and MCL13,respectively, in 0.01 M phosphate buffer (pH 7.5) and incubatedovernight at 4° C. As a positive control, plates were coated with 100 μgof purified cathepsin L in 0.05 M carbonate buffer pH 9.5, and incubatedovernight at 37° C. Between all incubation steps plates were washedthree times with 0.05% TWEEN®-80 in tap water. An additional blockingstep and drying off the plates was performed overnight by an “in-housemethod.” One hundred μg of calve or sheep serum, diluted 1/25 in 0.01 Mphosphate buffer (pH 7.5), containing 0.05% TWEEN®-80 and 0.5 M NaCl,were added for one hour at 37° C. HRPO-conjugated monoclonal antibodyagainst bovine IgG1 ( 1/30; ID-DLO, Lelystad, NL) and polyclonalanti-sheep IgG ( 1/15,000; Dakopatts) in 0.01 M phosphate buffer (pH7.5) containing 0.05% TWEEN®-80, 0.5M NaCl and 1% normal horse serum,were added for one hour at 37° C. Tetramethylbenzidine (0.005% H₂O₂ and1 mg/ml TMB in 0.1M Na-acetate/0.1 M citric acid buffer, pH 6.0) wasused as substrate. Five minutes after application of the substrate, thereaction was stopped with 0.5 M H₂SO₄, and extinctions were measured at450 nm in an Easyreader spectrophotometer (SLT, Vienne). The cut-offvalue between negative and positive was calculated as the average plusthree times the standard deviation of the OD 450 nm of sera fromparasite-free sheep or cows, respectively.

Results

Reproducibility of the Ex Vivo Gut Model

The accuracy of NEJ quantification in our infection and immunity modelwas tested in 18 rats. First, we determined the exact infection dose.After inoculation of an exact number of NEJs into a gut segment, NEJsremaining in needle and syringe were counted. This rest fraction,comprising on average 24% (range 6% to 56%) of the inoculation dose, wassubtracted from the inoculation dose. Six hours after infection wedetermined the peritoneal fraction, the luminal fraction and the gutfraction (using an immunohistochemical procedure) and the sum of thesefractions was compared with the infection dose (ranging from 4 to 78NEJs/cm). The peritoneal fraction ranged from 4 to 33 NEJs/cm (43% to80% of the infection dose, AVG 57%), the luminal fraction from 0 to 10NEJs/cm (0% to 6%, AVG 1%), and the gut fraction from 0.2 to 19 NEJs/cm(6% to 44%, AVG 32%). The mean total sum of NEJs recovered was 87%(±3.6% SEM) of the infection dose, demonstrating the grade ofreproducibility of the gut model.

Expression of Resistance

Infection with Fasciola hepatica results in resistance against achallenge at gut level.

Four weeks after oral infection with Fasciola hepatica rats were almostcompletely protected against a challenge infection. The number ofchallenge parasites that reached the liver of infected rats was reducedwith 97% (±1.1% SEM; n=13), as compared to naive rats.

A large part of resistance against Fasciola hepatica was expressed inthe gut mucosa, the porte d'entree of the parasite. Migration of NEJsthrough the intestinal wall of immune and naive rats was compared, usingthe ex vivo gut model. In immune rats resistance was expressed withintwo hours after challenge. After six hours, when migration wascompleted, 52% (±2.37% SEM; n=40) of the challenge NEJs had penetratedthe jejunum of naive rats, whereas in immune rats only 12% (±1.77% SEM;n=40) had traversed the gut wall. Thus, as a result of infection NEJmigration through the jejunum wall was reduced with 78%. Considerableresistance was also detected in the duodenum (50% reduction in NEJmigration), mid jejunum (65% reduction) and ileum (75% reduction). Thus,the entire small intestine is an important immune barrier. The durationof resistance was at least three months (n=6).

Induction of Resistance

To investigate the site in the host where resistance against Fasciolahepatica is induced, we followed the infection route of the parasite:gut mucosa-peritoneal cavity-liver.

The role of gut penetration in the induction of resistance wasinvestigated in the following way. After gut penetration of NEJs of theprimary infection, further migration of NEJs to the liver was preventedby 1) flukicide treatment of the rats or 2) capturing the NEJs using theex vivo gut model. Both flukicide treatment of the rats and capturing ofNEJs after gut penetration prevented further migration to the liver,because four weeks after infection, all rats had healthy looking livers.Surprisingly, none of the rats were protected against a challengeinfection. Thus, gut passage by itself does not induce resistanceagainst Fasciola hepatica expressed in the gut mucosa.

After penetration of the intestinal wall Fasciola hepatica enters theperitoneal cavity and migrates towards the liver. This route wasimitated by injecting NEJs of the primary infection in the peritonealcavity or between the liver lobes. As a result, four weeks afterinfection all rats were highly resistant against a challenge infection.The average level of protection at gut level was 78.8% (±4.6% SEM;n=11). Apparently, immunity is induced in the route peritonealcavity-liver and not during gut passage. Based on these results in latervaccination studies the antigen was injected in the peritoneal cavity.

Immune Mechanisms Against Fasciola hepatica in the Gut Mucosa

Gut segments of immune and naive rats were prepared for(immuno)histochemistry and compared for immunoglobulin, T cell, NK cell,goblet cell, macrophage, mucosal mast cell and granulocyte responses. Inimmune rats frequencies of mucosal mast cells, eosinophils andIgE-positive cells were significantly increased, as compared to naiverats. Upon re-infection of immune rats with Fasciola hepatica in asegment of the jejunum, challenge parasites are eliminated in the gutmucosa within two hours. At this time interval after infection challengeNEJs were coated with IgG1 and IgG2a antibodies. At the same timeinfiltrates of eosinophils were associated with the NEJs. Moreover, thelevel of protection at gut level strongly correlated with eosinophilresponses in the gut mucosa and IgG1 responses directed againstNEJ-antigen in the serum. These observations indicate that IgG1 (andIgG2a) antibodies and eosinophils are essential for protection.

Vaccination Studies

Stages of Fasciola hepatica

The developmental stage of Fasciola hepatica inducing the bestprotection was investigated. Extracts of NEJs and adult flukes wereprepared and injected intraperitoneally. Antigens from the NEJ stageappeared far superior: 57.3% (±6.2% SEM; n=10) protection at gut levelwas achieved, whereas adult stage antigens resulted in only 13.3% (±6.2%SEM; n=11) protection.

To measure the total level of protection induced by antigens from bothstages, challenge parasites reaching the target organ, the liver, wererecovered. Using NEJ antigen as vaccine almost complete protection wasachieved. The level of protection in these rats was 92.6% (±2.5% SEM;n=13). Adult stage antigens resulted in 56.3% (±15.9% SEM; n=8)protection.

Isolation of NEJ Antigen Fraction

Because immunoblot studies with sera from cattle and rats revealed twolow molecular weight (LMW) NEJ antigens only recognized by immune rats(>70% protection), a limited NEJ antigen fraction was isolated by meansof YM-30 filtration. During the procedure only 3% of the proteintraversed the YM-30 membrane and the number of antigens was reduced frommore than 50 to about five. Vaccination of rats with this LMW fractionresulted in 80% (±14% SEM; n=11) protection, based on the number ofparasites that reached the target organ, the liver. Of the eleven ratstested, six rats were 100% protected, and all this without the use ofany adjuvant! Also, at gut level, considerable resistance was expressed,54% (±12% SEM; n=7).

Identification of Vaccine Antigens

To identify the protective antigens present in the YM-30 filtrate,proteins were separated by SDS-PAGE. After electroblotting of theproteins onto a PVDF membrane different parts of the membrane were usedfor immunoblotting, protein staining and N-terminal sequence analysis,respectively. Protein staining revealed five protein bands withapproximate molecular weight of 30-32 kD, 28 kD, 25 kD, 20 kD and 12 kD,respectively. Of these proteins, only the 30-32 kD protein wasrecognized by all rats vaccinated with the YM-30 isolate (n=6), and wasclearly immunodominant. Together with the observation that in naturalimmune rats challenge NEJs are coated with IgG1 antibodies and that thelevel of IgG1 in the serum is strongly correlated with protection, weconclude that this 30-32 kD protein is a protective antigen. The 30-32kD protein was also recognized by orally infected rats (n=6), ratsvaccinated with NEJ extract (n=6) and orally infected calves (n=6). Onthe contrary, the antigen was not recognized by rats vaccinated withadult stage antigens (n=3).

Vise versa, on immunoblots of the YM-30 filtrate obtained from adultflukes, no reaction was observed with sera from the vaccinated rats,infected rats or infected cattle.

The 30-32 kD protein band was excised from the PVDF-membrane and furtheridentified using N-terminal sequencing. The protein displayed aN-terminal amino acid sequence comprising the sequence XXDVSWPFWDRMYNY(SEQ ID NO:1), in which the amino acids are given in the one lettercode.

The N-terminal amino acids of the 30-32 kD immunogen showed 69% homologywith the N-terminus of NEJ protein 4, as described by Tkalcevic et al.(1995), a 40 kD protein under non-reducing conditions. The N-terminus ofthe here disclosed 30-32 kD protein shows 54% homology with theprosequence of cathepsin L derived from adult F. hepatica (Wijffels etal. 1994). Characterization of the N-terminal of the 28 kD and 25 kDproteins from the PVDF membrane revealed the following sequences:

-   (i) XXWAVLVAGGSD (SEQ ID NO:17). This sequence shows 70% homology to    the N-terminus of NEJ haemoglobinase, according to Tkalcevic et al.    (1995)-   (ii) DVPASIDWRQYGYVTEVKDQ (SEQ ID NO: 18). This sequence is 95%    homologous to the N-terminus of NEJ cathepsin L according to    Tkalcevic et al. (1995) and 80% homologous to the N-terminus (aa 107    to 126) of mature cathepsin L according to Wijffels et al. (1994).

The immunoblotting studies together with the N-terminal sequenceanalyses demonstrate that procathepsin L is an immunodominant,protective antigen, whereas the enzymatic active cathepsin L is onlyoccasionally recognized by immune cattle or rats. We show here that thepresence of the prosequence (proregion) of cathepsin L is crucial forimmunogenicity and protection. Moreover, the studies indicate thatprocathepsin L derived from juvenile stages such as NEJs is moreprotective than procathepsin L derived from adult stages.

Vaccination of Rats with Synthetic Peptides Derived from Cathepsin L

Rats were vaccinated with two synthetic peptides derived from theprosequence of cathepsin L, MCF03 (aa 15 to 33) and MCF06 (aa 25 to 42),and with a peptide derived from the mature enzyme, MCF04 (aa 110 to129). When rats were vaccinated with peptide MCF03 or MCF06, these ratswere protected against a challenge infection (FIG. 1). The bestprotection was obtained when the peptides were conjugated to a carrierand applied in the presence of adjuvant. However, when rats werevaccinated with peptide MCF04, no protection against a challengeinfection was obtained. These results again support our finding that theprosequence (proregion) or fragments thereof of cathepsin L is crucialfor the induction of protection.

Amplification of a Unique Family of Cathepsin L Molecules from F.hepatica using NEJ-Specific Primers.

With primer set (i), the prosequence of cathepsin L was amplified, usingadult F. hepatica cDNA as template. Three positive clones (da16, da12and da13) were sequenced. Because of our primer choice, the clonesstarted at nucleic acid 85 (Trp 21) and ended at na 381 (Tyr 119),according to the sequence as Wijffels et al. (1994). The sequences ofthe propeptide parts of these clones showed 96.5% to 98.4% homology tothe sequence from Wijffels et al. (1994). The derived amino acidsequences showed 95.3% to 97.7% homology to the sequence of Wijffels etal. (1994).

With primer set (iii), the prosequence of cathepsin L was amplified,using cDNA from NEJs as template. Two PCR products were identified onagarose gel. The oligonucleotide with the expected size of 300 bp waspricked, amplified again using the same primers, and then ligated intothe TA cloning vector. Of five different clones, the nucleotidesequences were determined. Forward and reverse sequence analysisrevealed identical sequences. Clones da27, da26, da214 and da210 werevery homologous, having 90.3% to 98.4% identities. These clones showed79.5% to 82.2% homology to the prosequence (proregion) from Wijffels.The derived amino acid sequences showed 79.1% to 80.2% homology to thesequence from Wijffels. Clone da211 had a more different sequence andwas more homologous (87.4) to the prosequence of Wijffels. The derivedamino acid sequence showed 87.2% homology to the sequence from Wijffels.These data demonstrate that with the NEJ-specific primers a different“subfamily” of F. hepatica cathepsin L propeptides was amplified fromNEJS, compared with the products amplified from adult F. hepatica usingan adult F. hepatica specific primer set (16.6% to 23% discrepancies).

Moreover, the derived amino acid sequences from the “NEJ clones” reveala significant change in the site where the prosequence from cathepsin Lis cleaved off. In cathepsin L derived from adult F. hepatica, theprosequence is cleaved off between aa 106 and 107, with Arg at the P1position, the uncharged polar Asn at the P2 position and Ala at the P1′position. In the four homologous cathepsin L clones obtained with theNEJ-specific primers, however, we found Asn at the P1 position, Asp orGly at the P2 position and Asp at the P1′ position. It is possible thatother enzymes are needed to cleave off the propeptide of the “NEJcathepsin L.” This may result in a less efficient (auto)activation ofthe proprotein in the NEJ, compared with adult parasites. Since theprosequence is found essential for the induction of protection, thislikely explains the high levels of protection obtained with NEJantigens, compared with adult stage antigens. It may also explain theabsence of an immunoreactive proprotein in antigen extract from adult F.hepatica, as demonstrated on immunoblot.

With primer sets (ii) and (iv) the entire procathepsin L was amplifiedwith adult stage cDNA as template.

Cathepsin L Peptide ELISA for the Diagnosis of F. hepatica Infection inCattle and Sheep

Sera from two calves, sampled at regular intervals after infection withF. hepatica, were used to screen the peptide epitopes from cathepsin L(FIG. 4). No reactivity was observed with the peptide epitopes derivedfrom the prosequence of cathepsin L, MCF03 and MCF06, and only lowreactivity of one calve was detected with peptide MCL13. On thecontrary, both calves gave a strong reaction with peptide MCF05 andespecially peptide MCF04, from day 54 till at least day 154 afterinfection, comparable to the reaction obtained with purified cathepsinL. Combining of peptides MCF04 and MCF05 did not increaseimmunoreactivity. Accordingly, peptides MCF04 and MCF05 werespecifically recognized by sera from four sheep (ten weeks afterinfection with F. hepatica).

Sera from 24 calves, monoinfected with F. hepatica, and from calvesmonoinfected with other, relevant parasites, were tested in the ELISAwith peptide MCF04 and purified cathepsin L. All F. hepatica infectedcalves gave a positive reaction with both cathepsin L and peptide MCF04.On the contrary, neither of the calves infected with other, relevantparasites reacted with peptide MCF04. Accordingly, the D. Viviparusinfected calves did not recognize peptide MCF04, whereas they gave astrong reaction with cathepsin L (cross-reactivity).

Peptide MCF04 was recognized by F. hepatica infected sheep from week 5until at least week 16 after infection (FIG. 5). Panels of sera fromsheep, infected with other, relevant parasites were also tested in thepeptide ELISA (FIG. 5). Almost no reactivity of these sera with peptideMCF04 was detected.

These results demonstrate that an ELISA based on peptide MCF04 fromcathepsin L is both sensitive and specific. We conclude that this ELISAis highly valuable for diagnostic purposes regarding F. hepaticainfections, both for cattle and sheep. This peptide ELISA overcomes theproblem of cross-reactivity, especially found with D. Viviparus infectedcalves. Moreover, because naturally infected calves and sheep do notrecognize the protective peptide epitopes MCF03 and MCF06, thecombination of MCF04 for diagnostic purposes and peptides such asMCF03/MCF06 for vaccination purposes has considerable potential for avaccine.

PCR, Subcloning and Sequencing

To further study and obtain the isolated nucleotide sequence of aprotective protein useful for vaccination against a wide range ofparasitic infections, amplification, cloning and sequencing techniquesknown in the art are used. For example, in the case of the protective30-32 kD protein of Fasciola hepatica, in a first step in RT-PCR,primers A and B are used. The sequence of primer A involves a set ofdegenerate oligonucleotides deduced from the N-terminal amino acidsequence. Primer B is, for example, deduced from a spliced leadersequence located upstream at the 5′ end of parasitic mRNA (Davis et al.,The Journal of Biological Chemistry, 31: 20026-20030, 1994). Afteramplification the obtained fragments are cloned and sequenced. A primerC is than selected located in the sequence between A and B and usedtogether with a poly (dT) primer to amplify the corresponding 3′ part ofthe wanted nucleotide sequence, after which the whole gene or selectedfragments thereof are cloned and sequenced. By using the isolatednucleotide sequences in amplification or screening methods varying ordifferent parasitic sequences that encode functionally equivalentproteins can be identified in and isolated from related parasites. Allsuch nucleotide sequences or fragments thereof can be cloned by methodknown in the art in suitable expression systems to generate recombinantproteins that can be used in anti-parasite vaccines or for diagnosticpurposes, as described above. Assessment of the protective value of suchproteins can of course be performed in the ex vivo model provided by theinvention. The nucleotide sequences alone, or incorporated in suitablevector systems or constructs can also be employed in DNA vaccinationprotocols.

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1. An isolated or recombinant protective protein derived from aproregion of a cathepsin-like protease that is specific for a newlyexcysted juvenile (NEJ) stage of Fasciola hepatica wherein the proteincomprises the amino acid sequence selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:24, and SEQ ID NO:26.
 2. The isolated orrecombinant protective protein according to claim 1, as shown to beprotective in a method comprising using an ex vivo animal or challengemodel to measure protective immunity directed against a parasite.
 3. Theprotective protein of claim 1, wherein said protein is a recombinantprotein.
 4. A vaccine comprising the protective protein of claim
 1. 5.The vaccine of claim 4 further comprising a pharmaceutically acceptablevehicle.
 6. The vaccine composition of claim 5, wherein said protectiveprotein is recombinant.
 7. A diagnostic test comprising: the protectiveprotein of claim 1 and a molecule to detect immune complexes between theprotective protein of claim 1 and antibodies from a sample of a subjectto be tested.
 8. A method for immunizing an animal against infection byFasciola hepatica, the method comprising administering to the animal thevaccine composition of claim
 5. 9. The method according to claim 8,wherein Fasciola hepatica is of the type that infects a host through amucosal or skin surface.